Tcr/mhcii-collagen interaction inhibitors useful for the treatment of rheumatoid arthritis

ABSTRACT

The T cells specific to human collagen type II, one of the possible autoantigens, have a crucial role in the development of rheumatoid arthritis in the context of HLA-DR4. The protein-protein interactions between the T cell receptor (TCR) and the type II collagen linked to the allele MHC of class II HLA-DR4 may thus represent the target for the development of new drugs against rheumatoid arthritis. Using computational virtual screening techniques, families of pharmacologically active molecules have been identified that interfere with the TCR/collagen ll-MHCII interaction. The compounds identified here open up new possibilities in the treatment of rheumatoid arthritis.

STATE OF THE PRIOR ART

Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects0.5-1% of the population. It is a progressive disease that leads to thefunctional impotence of many joints. The etiology of the disease isunknown, but autoimmunity plays a dominant role in its pathogenesis. Infact, T cells and auto-antibodies are present in the blood of patientswith RA. Most of the autoreactive T cells are specific to human collagenand can be found in inflamed joints where they promote the inflammatoryresponse which ultimately leads to the destruction of cartilage. Asconfirmation of the role of T cells, RA has been demonstrated to beassociated with the alleles HLA-DR 1 and 4. The present inventors havereported in the literature that it is possible to find T cells that arespecific to human collagen type II in the context of HLA-DR4 in theblood and synovial fluid of patients, and the level of cells in theblood varies in parallel with the activity of the disease (Ria et al,Arth Res Ther 2008, 10: R135). Indeed, we have reported that the T cellsspecific to the peptide 261-273 of collagen type II (huCollp261) arepresent in large numbers in the blood at the onset of the disease,decreasing during the phases of clinical remission and increasing againat the time of relapses. A portion of these cells is spontaneouslyenriched in the synovial fluid during synovitis. From a methodologicalpoint of view, the method used allows to identify specific individual Tcells through the identification of the sequence of the most variableregion of the T cell receptor (TCR), the CDR3 region of the beta chainof the TCR, by PCR. The TCR recognizes the antigen to which it isspecific as a complex of a peptide (in general comprised of lengthbetween 8 and 15 amino acids) with a molecule called HLA (“the peptideis presented by the HLA molecule to the TCR”). Each individual hasbetween 12 and 20 of these molecules, each having its own chemicalcharacteristics and each capable of binding a group of peptides whichshare the presence, in certain positions, of some amino acids. Each ofthe HLA molecules (which are coded in 6 different and contiguous geneloci on chromosome 6) has very wide genetic variability, with hundredsof alleles. The set of HLA molecules of an individual is called ahaplotype. As mentioned above, in RA, 50% of patients have HLA-DR4 orDR1 in their haplotype. Both of these HLA molecules effectively presentthe peptide huCollp261. It should be added that the allele HLA-DR7 alsopresents the same type of peptides presented by HLA-DR4 and DR1. Weestimate that about 70% of the T cells identified by the immunoscopetechnique in each patient are effectively specific to the complexDR4/huCollp261, on the basis of the fact that this is the fraction ofcells that actually shares the use of common TCRs, this is thepercentage of cells that proliferate only in response to this peptideand not in response to other control peptides (Ria et al Arth Res Ther2008) and also based on the observation that this is the percentage ofcells able to recognize this peptide in the context of anothercross-reactive protein (unpublished observations). It should behighlighted, on the other hand, that also patients that are not positivefor HLA-DR4, 1 or 7, may sporadically have the presence of cells thatproliferate in response to the peptide huCollp261, but in this case thisresponse is not related to the disease state. The use of the TCRsequences in the diagnosis and treatment of RA in these subjects wasalso the subject of a previous patent by F. Ria and G. F. Ferraccioli(RM2007A000429 and international PCT: PCT/IB2008/053152). The presentinventors have subsequently described the three-dimensional interactionbetween the beta chain of the TCR and the complex formed by the humancollagen peptide 261-273 (huCollp261) with HLA-DR4 (De Rosa et al,PloSOne 2010, 5:e11550), and this work forms the basis of the object ofthe present patent application. Since the etiology of RA is unknown,therapy has focused on the common pathogenic mechanism, i.e.,inflammation. The current protocols of therapy include different typesof medications: NSAIDs (nonsteroidal anti-inflammatory drugs), steroidsand so-called DMARDs (disease-modifying anti-rheumatic drugs) which alsoinclude biological response modifiers, monoclonal antibodies capable ofblocking TNF-α and IL-1, the major cytokines involved in theinflammatory response. Treatment in most cases includes the use of morethan one of these drugs. Although therapy is effective in decreasing theseverity of the disease, in the majority of cases the progressioncontinues. In this regard, see the guidelines of the Italian Society ofRheumatology in Clin Exp Rheumatol. 2011 (3 Suppl 66):S7-14. —Clin ExpRheumatol. 2011 (3 Suppl 66):S15-27. —Clin Exp Rheumatol. 2011 (3 Suppl66):S42-62). In addition, these drugs can have serious side effects,generally proportional to their effectiveness, and often the treatmentis followed by immunosuppression that allows the development ofopportunistic infections or reactivation of latent infections that causediseases such as tuberculosis and progressive multifocalleukoencephalopathy. Therefore there exists the need for new drugs thatinterfere more specifically with autoimmune response mechanisms, butthat maintain the ability to control infectious agents.

Following an idea originally proposed in 1988 by L. Adorini, Liu Z, LiB, Li X, Zhang L, Lai L. published an article on the identification ofmolecules that inhibit the HLA-DR4-peptide interaction, entitled“Identification of small-molecule inhibitors against human leukocyteantigen-death receptor 4 (HLA-DR4) through a comprehensive strategy” J.Chem Inf Model. 2011 Feb. 28; 51(2):326-34. In this work scaffolds areidentified for compounds capable of occupying the pocket of the HLA-DR4molecule with greater affinity than other peptides that can be used forthe same purpose. In order to apply this approach to RA therapy, themajor limitation of these products is the need to block all HLA-DR4molecules in order to obtain the desired therapeutic effect. Theaforementioned article is irrelevant for the purposes of the presentpatent, since it has advantages of selectivity of the target and doesnot interfere with the recognition of the collagen by the T cells, butrather with the presentation of peptides in the context of HLA-DR4. Infact, to achieve a therapeutic effect with the products identified byLiu et al. the activity is also inhibited of all those T cells thatrecognize other peptides, derived from pathogens in complex withHLA-DR4. The objective of the product that we are presenting is to focustherapeutic intervention only on these complexes, thereby gainingadvantages in terms of specificity, selectivity and pharmacologicalfeasibility.

BRIEF DESCRIPTION OF THE INVENTION

The T cells specific to human collagen type II, one of the possibleautoantigens, have a crucial role in the development of rheumatoidarthritis in the context of HLA-DR4. The protein-protein interactionsbetween the T cell receptor (TCR) and the type II collagen linked to theallele MHC of class II HLA-DR4 thus represent the target for thedevelopment of new drugs against rheumatoid arthritis.

The present invention arises from the search for compounds capable ofinhibiting the proliferation of T cells as a result of the interactionbetween the T cell receptor (TCR) and the type II collagen linked toallele HLA class II. Using PBMCs (Peripheral Blood Mononuclear Cells) ofpatients suffering from rheumatoid arthritis the possible toxicity ofcandidate inhibitors is first examined in vitro and subsequently, theability is demonstrated of these inhibitors to specifically block theresponse to human collagen in the context of HLA-DR4 and the similarHLA-DR7.

Thus a family of molecules is identified that is able to selectivelyblock only the immune response to the autoantigen, leaving the rest ofthe immune system intact and efficient.

Therefore the object of the invention are compounds with inhibitingactivity of the interaction between the TCRs and the MHCII-Humancollagen type II complex, or the MHCII complex and the antigenic peptidefragment in position 261-273 of human collagen type II (huCollp261),while they are irrelevant to the remaining functions of the immunesystem.

In particular the object of the present application are compounds havingthe general formula selected from the formulas (I), (II) and (III) foruse in the treatment of RA.

Making up part of the compounds of formula (I) is the new compound9-[4-(3(R)-phenyl-1(S)-propilpirrolidin-1-io-1-yl)but-2-ynyl]fluoren-9-ol and itsanalogues. Making up part of the compounds of formula (II) is thecompound:(E)-3-(3,6-dichloro-9H-carbazol-9-yl)-N-(4-hydroxy-benzylidene)propanidrazideand compounds of formula (III) the compoundN1,N5-bis(dibenzo[b,d]furan-3-il)glutarammide (formula III).

The compounds of formula (II) and (III) have less inhibition activitythan the compounds of formula (I), but are nevertheless usable intherapy.

Among these compounds, some have been synthesized for the first time bythe present inventors. Therefore a second object of the invention arenew compounds that fall under the generic formulas (I), (II) and (III).

A third object of the invention are the pharmaceutical compositionscontaining all of the compounds of formula (I), (II) and (III) andpharmaceutically acceptable excipients.

A fourth object of the invention is a method for preparing moreeffective compounds, of formula (I).

The compounds of the invention can be used advantageously in thetherapeutic treatment, including symptomatic, of rheumatoid arthritis.

DESCRIPTION OF FIGURES

FIG. 1: The figure shows the results of a T cell proliferation teststimulated by the antigenic peptide 261-273 from human collagen(huCollp261). The test is performed in the absence of antigen (ag−), inthe presence of antigen (ag+) and in the presence of ag+ and of acandidate inhibitor compound (2S,1S and 3S). The compound 2S, i.e.(9-[4-(3(R)-phenyl-1(S)-propilpirrolidin-1-io-1-yl)but-2-ynyl]fluoren-9-ol)is capable of strongly depressing (about 100° A) the RelativeStimulation Index (RSI) in the context of HLA-DR4 and HLA-DR7. Thecompound 1S (i.e.(E)-3-(3,6-dichloro-9H-carbazol-9-yl)-N′-(4-hydroxy-benzylidene)propanidrazide) strongly depresses (about 100%) the RSI in the contextof HLA-DR11, while the compounds 2S and 3S, i.e.,(N1,N5-bis(dibenzo[b,d]furan-3-yl) glutarammide), depress RSI lesseffectively, but nevertheless significantly (almost 20%), in the contextof HLA-DR11.

FIG. 2: The figure shows the results of a T cell proliferation testobtained from a patient DR1+, stimulated by the antigenic peptide261-273 from human collagen (huCollp261). The test is performed in theabsence of antigen (ag−), in the presence of antigen (ag+) and in thepresence of ag+ and of a candidate inhibitor compound (2S,1S).

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention share the same pharmacological activity,namely the inhibition of the interaction between the TCRs and theMHCII-Human collagen type II complex, or the MHCII complex and theantigenic peptide fragment in position 261-273 of human collagen type II(huCollp261), despite being irrelevant to the remaining functions of theimmune system.

Chemically the derivatives of the invention are molecules that sharesome biologically equivalent chemical structures such as the fluorinegroup, the carbazole group, dibenzofuran group and having the generalformula selected from between formula (I), (II) and (III).

In particular the object of the invention are derivatives of 9-hydroxyfluorene of general formula (I)

where R is a substituent selected from hydrogen, C1-C5 linear orbranched alkyl, aryl-C1-C5-alkyl including halogen substituted on thephenyl group, and R¹ is a substituent selected from phenyl, halogenortho-, meta- or para-substituted phenyl, where, in all cases, halogenis selected from chlorine, bromine or iodine, or R¹ is a group 2′- or3′- or 4′-pyridyl.

In one embodiment of the invention, R is selected from hydrogen, amethyl group, ethyl, linear or branched propyl, linear or branchedbutyl, phenylethyl including substituted halogen ortho, for exampleo-chloro or o-iodo. R1 is selected among the groups: phenyl, 2′-pyridyl,3′-pyridyl, 4′-pyridyl, o-iodophenyl, m-iodophenyl, p-iodophenyl,o-chlorophenyl, m-chlorophenyl, p-chlorophenyl.

Specific compounds of formula (I) are the compounds 1 to 15 below.

Among these, compound 1 is9-[4-(3-phenyl-1-propilpirrolidin-1-io-1-yl)but-2-ynyl]fluoren-9-ol, inthe form of a diastereoisomeric mixture.

All the compounds of formula (I) comprise two centers of asymmetry inpositions 1 and 3 of the pyrrolidine group. An integral part of thepresent invention are diastereoisomeric mixtures (RR)/(SS) and (RS)/(SR)or individual racemic mixtures or individual enantiomers (RS), (SR),(RR), (SS). All the compounds of formula (I) may be in the form ofammonium salts, for example chloride, bromide, iodide.

The table below shows the binding affinity of the values of theindividual stereoisomers of9-[4-(3-phenyl-1-propilpirrolidin-1-io-1-yl)but-2-ynyl]fluoren-9-olcalculated by the molecular docking program AutoDock (Morris et al. J.Computational Chemistry, 19:1639-1662, 1998). These values are providedby the “scoring” function of the program. The docking process consistsin virtually reproducing the association between a protein and a ligandby selecting the energetically favored complex, i.e. the one with thelowest interaction energy value (kcal/mol). The docking programs arecharacterized by the type of algorithm used and by the “scoring”function used. In a virtual screening the scoring functions are used toselect the best conformation of the molecule within the site and, at theend of the process, to estimate the binding affinity of the complexesformed by the various candidate ligands.

Compound Binding affinity (kcal/mol)9-[4-(3(R)-phenyl-1(S)-propilpirrolidin-1- −8.52io-1-yl)but-2-inyl]fluoren-9-ol9-[4-(3(S)-phenyl-1(S)-propilpirrolidin-1- −5.92io-1-yl)but-2-inyl]fluoren-9-ol9-[4-(3(S)-phenyl-1(R)-propilpirrolidin-1- −7.26io-1-yl)but-2-inyl]fluoren-9-ol9-[4-(3(R)-phenyl-1(R)-propilpirrolidin-1- −6.98io-1-yl)but-2-inyl]fluoren-9-ol

In particular, the preferred isomer of the compound 1 of Figure (I) is9-[4-(3(R)-phenyl-1(S)-propilpirrolidin-1-io-1-yl)but-2-ynyl]fluoren-9-ol,which has the highest binding affinity, of all the compounds analyzed,to the T cell receptor (TCR) as highlighted by the lowest interactionenergy (−8.52 kcal/mol).

Below are the binding affinities for all compounds of formula (I)calculated by the program AutoDock.

Compounds of formula (I) Binding affinity Compound (kcal/mol) 1 −8.52 2−8.37 3 −8.31 4 −8.27 5 −8.09 6 −7.59 7 −8.21 8 −6.67 9 −8.47 10 −6.3311 −7.48 12 −6.17 13 −8.51 14 −7.66 15 −7.24

In an alternative embodiment of the compounds of the invention arecompounds with formula (II),

in which R is a substituent selected from hydrogen and halogen, selectedfrom chlorine, bromine and iodine; R¹ is a substituent selected fromhydrogen or halogen selected from chlorine, bromine or iodine; R² is asubstituent selected from hydroxyl or C1-3 alkyl (methyl; ethyl,n-propyl, isopropyl); R³ is a group selected from —N═ and —CH═ and R⁴ isa group selected from N or CH.

The preferred compounds of formula (II) are the compounds 1 to 15 below,including the compound(E)-3-(3,6-dichloro-9H-carbazol-9-yl)-N′-(4-hydroxy-benzylidene)propanidrazide (compound 1).

All the compounds of formula (II) may be in salt form on one of thenitrogen atoms, such as for example chloride, bromide, iodide.

In the case where R⁴ is “═CH-” and R and R¹ are different, R⁴ is acenter of asymmetry. In this case, both the racemic mixture and theindividual enantiomers (R) or (S) that separate a mixture enriched withone of the enantiomers are part of the invention.

Below are the binding affinities for all compounds of formula (II)calculated by the program AutoDock.

Compounds of formula (II) Binding affinity Compound (kcal/mol) 1 −7.82 2−5.57 3 −6.34 4 −7.59 5 −7.44 6 −7.23 7 −7.59 8 −7.76 9 −7.80 10 −6.2411 −5.83 12 −7.29 13 −7.23 14 −6.01 15 −6.07

In an alternative embodiment of the compounds of the invention arecompounds with formula (III),

in which R is a substituent selected from hydrogen and halogen, selectedfrom chlorine, bromine and iodine; R¹ is a substituent selected fromhydrogen or halogen, selected from chlorine, bromine or iodine; R² isselected from O, NH, CH₂; R³ is selected from O and S.

All the compounds of formula (III) may be in salt form on one of thenitrogen atoms, for example chloride, bromide, iodide. The preferredcompounds of formula (III) are the compounds 1 to 15 below. The compound(1) is N1,N5-bis(dibenzo[b,d]furan-3-yl) glutarammide.

Below are the binding affinities for all compounds of formula (III)calculated by the program AutoDock.

Compounds of formula (III) Binding affinity Compound (kcal/mol) 1 −8.202 −6.89 3 −6.54 4 −8.12 5 −7.61 6 −6.43 7 −6.41 8 −6.53 9 −6.42 10 −7.4711 −6.43 12 −6.90 13 −7.96 14 −7.56 15 −7.53

The compounds of the invention are identified and selected the firsttime through a “structure-based drug design” approach based on thethree-dimensional structure of the ternary complexTCR/huColl261/HLA-DR4.

However, some of the compounds of the invention are commerciallyavailable through appropriate producers. In particular, the compound ofgeneral formula (I) molecule 1 can be purchased from the sitewww.ibscreen.com using the identification code: STOCK2S-15693, molecules2-5 can be purchased online from the site www.molport.com using therespective identification codes: MolPort-001-993-760,MolPort-001-991-570, MolPort-002-558-653, MolPort-002-559-198.

For the compound of general formula (II), molecules 1, 4 and 8 can bepurchased online from the site www.ibscreen.com using the respectiveidentification codes: STOCK1S-19103, STOCK1S-01057 and STOCK3S-37745.

For the compound of general formula (III), molecule 1 can be purchasedfrom the site www.ibscreen.com using the identification code:STOCK3S-02896.

All the known molecules can also be purchased from the companies:www.aurorafinechemicals.com, www.chemir.com, www.cyanta.com.

Unlike the previous compounds, compounds 6 to 15 of the General formula(I), compounds 2 and 3, from 5 to 7, from 9 to 15 of the General formula(II) and the compounds 2 to 15 of the general formula (III): are newcompounds, synthesized by the present inventors. These compounds assuch, their diastereoisomeric mixtures or conformational or racemicmixtures or isolated individual enantiomers, or their correspondingsalts, are a further object of the present application.

All the compounds of formula (I) can be reproduced using the synthesismethod shown schematically and described in example 1, which consists ofsteps and reactions that are in themselves well known and described inthe literature. This method is exemplified in relation to the specificcompound (1) in Formula (I), namely9-[4-(3-phenyl-1-propilpirrolidin-1-io-1-yl)but-2-inyl]fluoren-9-ol.

All the compounds listed in Formula (I) can be synthesized by the samemethod using the corresponding acceptable intermediates.

In the case of the compounds of formula (I), the preparation method mayinclude the additional step of resolution of the diastereoisomericmixture (RR)/(SS) and (RS)/(SR) in the individual racemic mixtures orthe separation of the individual enantiomers (RR), (SS), (RS) or (SR).

The separation can be performed using methods known to the expert, inwhich the separation is performed by zone electrophoresis (CapillaryZone Electropheresis CZE) using chiral selectors such as cyclo-dextrin.

The compounds of general formula (II) and (III) can also be synthesizedaccording to methods known to the expert.

The object of the invention are also pharmaceutical compositions whichcontain one or more compounds of the invention as their activeingredient together with a pharmaceutically acceptable excipient and,optionally, common additives and stabilizers in the pharmaceuticalindustry.

Such compositions are suitable for local or systemic use and may be bothin liquid formulation, solid, semi-solid, or suppository formulation.

Examples of liquid formulations are solution, suspension, emulsion,suitable e.g. for parenteral administration, local parenteraladministration, oral administration or also in spray form. Examples ofsolid formulations are tablets, dragées, capsules, granules,freeze-dried tablets, that are suitable for oral administration.Examples of semi-solid formulations are pastes, salves, gels, ointments,that are suitable for topical application.

Each of these formulations shall contain a quantity of the activecompound varying between 10 micrograms and 1000 mg, preferably between100 micrograms and 100 mg, or between 100 micrograms and 50 mg, forexample between 420 micrograms to 42 milligrams, for example 0.5, 1, 10,30 mg per dosage unit (based on the concentrations used in toxicitytests).

Furthermore, the object of the present invention is also a method forpreparing compounds of formula (II) comprising at least one of the stepslisted in the following scheme 2

The experimental work underlying the present invention has allowed forthe selection of molecules capable of partially inhibiting or totallyblocking the proliferation of T cells caused by the presence of thecorresponding antigen: in this case caused by human collagen type II,and more precisely its antigenic fragments, such as the fragment 261-273(huCollp261). Partial inhibition is defined as any degree of inhibitionless than 100%, between 10% and 99%, e.g. 20%, 30%, 40%, 50%, 60% 70%,80%.

The relevant observations are shown in FIG. 1 and Table I in example 2.In the experiment shown in FIG. 1, PBMCs from four patients (one DR4+,one DR7+ and two negative for the HLA-DR alleles associated with RA) arecultured in the absence or presence of huCollp261 (10 microM) and in thelatter case also in the presence of each of the three candidateinhibitors (I)-1 (2S), (II)-1 (1S) and (III)-1 (3S) at the sameconcentration. The results clearly indicate how 2S is able to block theproliferation of the cells that respond to huCollp261 in DR4+ or DR7+subjects. In agreement with the observation that these compounds arefree of toxic effects at concentrations up to 100 microM, theproliferation of sporadic T cells that respond to the same peptide, butin the context of HLA molecules different from DR4 1 or 7, remainsintact. In the experiment shown in Table I, we examined the ability of1S, 2S and 3S to inhibit the proliferation of T cells specific tohuCollp261 in a greater number of DR4 patients. In total, we examined 4DR4+ patients and observed the responses of 15 different T cells. Thecompound 1S inhibits 13% of these cells, 2S inhibits 60% and 3S inhibits45%, indicating that all three compounds possess inhibitory activity. Itis important to note that the three compounds have inhibitory activityon the same group of T cells; in other words the cells inhibited by 1Sare also inhibited by 2S and 3S, and the cells inhibited by 3S are alsoinhibited by 2S. This indicates that all three compounds have the samemechanism of action although they are not structurally identical.Finally it is stressed that the cells inhibited by 2S are also the onescapable of cross-recognition of the peptide (see above).

Without wishing to limit the invention to specific scientific theory, itis believed that the inhibition of T cell proliferation by the compoundsof the invention is the consequence of inhibitory interference on themechanisms of interaction between T cell receptors (TCR) and the HLADR-Collagen complex.

This hypothesis is supported by the following observations: 1. Thecompounds do not exhibit toxicity on PBMCs, 2. Although the compoundsare not structurally identical, they inhibit the proliferation of cellsfunctionally belonging to the same group, indicating that the mechanismthat underlies the inhibition is the same; 3. The T cells that aresubject to the inhibitory action are the same that have the ability tocross-recognize the peptide of other origin. Therefore the action of thecompounds is specifically limited to a homogeneous group of T cells thatshare the molecular basis of recognition of the complex huCollp261 incomplex with HLA-DR4, DR1 and DR7. In addition, the molecular modelingdata do not convincingly demonstrate that the compounds act by blockingthe binding pocket of the HLA molecule for the peptide huCollp261.

Therefore it can be reasonably stated that the molecules of theinvention selectively block only the immune response of theautoantigene, i.e. peptide 261-273 of Human type II collagen, leavingthe rest of the immune system intact and efficient.

The concentrations of the compounds tested for toxicity are of the orderof micromoles/l. For example, 1, 5, 10, 20, 50, 100 micromoles/l. Theconcentration tested for the activity is about 10 micromoles/l.

The experimental results demonstrate that the molecule (2S):9-[4-(3-phenyl-1-propilpirrolidin-1-io-1-yl)-but-2-inyl]fluoren-9-olcompletely blocks the proliferation of T cells specific to the collagenfragment huCollp261 in the context of DR4 (FIG. 1), that the molecule(1S): (E)-3-(3,6-dichloro-9H-carbazol-9-il)-N-(4-hydroxy-benzylidene)propanidrazide) strongly depresses RSI (about 100%) in the context ofHLA-DR11, while the compounds 2S (above) and 3S:N1,N5-bis(dibenzo[b,d]furan-3-yl) glutarammide, suppress theproliferation of T cells in the context of DR11 in a statisticallysignificant manner, albeit less effectively.

The invention will be illustrated below in detail in the followingexamples, which are by way of example, but not limiting.

EXAMPLES Example 1 Synthesis of compound9-[4-(3(S)-phenyl-1(S)-propilpirrolidin-1-io-1-yl)but-2-inyl]fluoren-9-ol.The synthesis is carried out according to scheme 1 below

I) The compound 1a (3 g, 1.66·10⁻² mol), commercially available, isdissolved in Et₂O (diethyl ether) (250 ml). To it is added an ethersolution of t-BuPh₂SiOCH₂CCCH₂MgBr (bromide of{4-[(tert-Butyldiphenylsilyl)-oxy]-2-butyn-1-yl}magnesium), (1.66·10⁻²mol in 250 ml) (Tetrahedon Letters 1993, 5467).

II) The solution of NH₄Cl (ammonium chloride) is added, stirred for 15minutes and then extracted with AcOEt (ethyl acetate). The organic phaseis concentrated under vacuum and purified by gravity chromatography[silica gel, CHCl₃(chloroform)-MeOH(methanol) (98:2)]. Product 1b isobtained with a yield of 70%.

III) To a solution of 1b (5.67 g, 1.162·10⁻² mol) and PPTS (pyridiniump-toluenesulfonate) (0.117 g, 4.6·10⁻⁴ mol) in CH₂Cl₂(dichloromethane)(500 ml), a solution of DHP (dihydropyran) (3.2 ml, 3.5·10⁻² mol) isadded dropwise. The reaction is left under stirring at room temperaturefor 4 h, washed with a saturated solution of NaHCO₃ (sodium bicarbonate)and extracted with CH₂Cl₂.

The organic phase is anhydrified with Na₂SO₄ (sodium sulfate), filteredand concentrated under vacuum.

IV) The crude is dissolved in anhydrous THF (tetrahydrofuran). Thissolution is brought to a temperature of 0° C., TBAF (tetrabutylammoniumfluoride) is added (2.63 g, 1.3·10⁻² mol) and it is put under stirringat room temperature for 45 min. The resulting mixture is diluted withCH₂Cl₂ and quenched with H₂O (water).

The organic phase is washed with brine (saturated solution of sodiumchloride), anhydrifed with Na₂SO₄ and concentrated under vacuum.

V) To a solution of compound 1c (3.88 g, 1.162·10⁻² mol) and Et₃N(triethylamine) (4.8 ml, 3.2·10⁻² mol) in anydrous THF (500 ml), PBr3(phosphorus tribromide) is added (1.5 ml, 1.162·10⁻² mol) at atemperature of −20° C. The reaction is left under stirring at roomtemperature for 4 h after which H₂O is added. The mixture thus obtainedis extracted with AcOEt. The organic phase is washed with brine(saturated solution of sodium chloride), anhydrifed with Na₂SO₄ andconcentrated under vacuum. The crude is purified by gravitychromatography [silica gel, Hexane-EtOAc (70:30)]. Product 1d isobtained with a yield of 50%.

VI) To a solution of compound 1d (2.3 g, 5.8·10⁻³ mol) and Et₃N (0.725ml, 1.16·10⁻² mol) in anydrous THF (380 ml), compound 3 (0.848 g,5.8·10⁻³ mol) is added (Khimiko-Farmatsevticheskii Zhurnal, 25(4), 60-2;1991) at a temperature of −20° C. The reaction is left under stirring atroom temperature for 4 h after which H₂O is added. The mixture thusobtained is extracted with AcOEt. The organic phase is washed with brine(saturated solution of sodium chloride), anhydrifed with Na₂SO₄ andconcentrated under vacuum.

VII) The crude is dissolved in hot THF and to the solution is addedn-PrBr (0.528 ml, 5.8·10⁻³ mol) (propyl bromide). The reaction is leftto reflux for 2 h and concentrated under vacuum.

VIII) The crude is solubilized in THFdry, the solution is brought to 0°C., acidified with AcOH 1M (acetic acid) and left under stirring for 3 hat room temperature. The solution is concentrated under vacuum.

Example 2 Synthesis of compound((E)-3-(3,6-dichloro-9H-carbazol-9-yl)-N′-(-4-hydroxy-benzylidene)propanidrazide

I) A solution of 3,6-dichloro-9H-carbazole (compound 1a), commerciallyavailable, (1.06 g, 4.5 mmol) and methyl acrylate (0.45 mL, 5 mmol) inacetic acid is left under stirring for 24 hours in a closed tube at atemperature of 90° C. The reaction mixture is purified in water and ice,basified with Na2CO3 and extracted in dichloromethane. The organicextracts are washed with a saturated solution of Na2CO3, dried overNa2SO4, and concentrated at reduced pressure. The residue is purified byflash chromatography (SiO2, eluting in gradient from hexane tohexane/EtOAc 8:2) to yield compound 1 b,methyl3-(3,6-dichloro-carbazol-9-yl) propionate (1.28 g, 88%).

II) A solution of 1 b (1.28 g, 3.96 mmol and hydrazine hydrate sol. 50%(0.25 mL, 4 mmol) in ethanol is heated to reflux and left under stirringfor 6 hours. The solvent is removed at reduced pressure. The compound1c, 3-(3,6-dichloro-carbazol-9-yl)-propionic acid hydrazide is obtainedin quantitative yield without further purification.

III) A solution of 1c (1.28 g, 3.96 mmol) e 4-hydroxybenzaldehyde (484mg, 3.96 mmol) in ethanol is heated to reflux and stirred for 12 hours.Then the solvent is removed at reduced pressure. The reaction crude isredissolved in ethyl acetate and washed with HCl 1N and with brine(saturated aqueous solution of NaCl). The organic phase is dried overNa2SO4, and concentrated at reduced pressure. The residue iscrystallized from a mixture of EtOAc/petroleum ether 40:60 to give acrystalline solid of compound 1 d, (4-hydroxy-benzylidene)-hydrazide(E)-3-(3,6-dichloro-carbazol-9-yl)-propionic acid (1.5 g, 90%).

Analogous synthetic procedure can be used for the compounds with formula(II) 2,3,4, using 3,6-dibromo-9H-carbazole, 3,6-diiodo-9H-carbazole and9H-carbazole as starting product, respectively.

Example 3 Synthesis of the compound 3S(N1,N5-bis(dibenzo[b,d]furan-3-yl) glutarammide

I) In a flask are introduced 366 mg of 1a (3-amminodibenzofuran, 2.0mmol), 1 b (132 mg of glutaric acid, 1.0 mmol), 135 mg of HOBT (1.0mmol), 10 ml of dichloromethane and 0:14 ml of triethylamine (1.0 mmol).The solution is cooled to 0° C. with an ice bath and 206 mg of DCC isadded (1.0 mmol). It is left under stirring for 15 minutes and then theice bath is removed. After a short time a white precipitate ofcyclohexylurea forms, it is left under stirring for another 3 h, thenfiltered on gooch and the solvent is evaporated at reduced pressure. Thecrude is purified by column chromatography (eluent EtOAc: hexane 25:75),the pure product 1c is obtained with a yield of 80%.

For the synthesis of the other derivatives it is sufficient to work inexcess of glutaric acid in such a way as to isolate the mono-amidifiedproduct. Then we proceed with the next reaction in the same conditionsbut using a 3-amminodibenzofuran or an appropriately substituted3-aminodibenzothiophene.

Example 4 T Cell Proliferation Test for DR4+ and DR7+ Patients

Mononuclear cells (PBMCs, Peripheral Blood Mononuclear Cells) areisolated from four patients with rheumatoid arthritis using a densitygradient. One patient is DR4+, one DR7+(DR4, DR7 and DR1 present thesame peptides and the response to human collagen peptide 261-273restricted by these alleles use the same TCRs); two patients arenegative for DR4, DR1 or DR7. The PBMCs are cultured at a concentrationof 5×106 cells/ml in 24-well plates in RPMI 1640 medium supplemented asdescribed (Arth Res Ther, 2008) without (ag−) or with the antigenicpeptide (10 μg/ml, ag+) or with the same concentration of peptide in thepresence of an equimolar concentration of the commercialdiastereoisomeric mixture of9-[4-(3-phenyl-1-propylpyrrolidin-1-ium-1-yl)but-2-ynyl]fluoren-9-ol(2S) comprising the active enantiomer9-[4-(3(R)-phenyl-1(S)-propylpyrrolidin-1-ium-1-yl)but-2-ynyl]fluoren-9-ol,in a final volume of 1 ml. After three days, the cells are collected andthe “immunoscope” analysis is performed as previously described. Thisanalysis allows for the identification of T cells that proliferate inresponse to the antigen. This proliferation is referred to as RSI(specific stimulation index) with a value of 1 corresponding to thenon-stimulated sample. The graph (FIG. 1) shows the ability of9-[4-(3-phenyl-1-propilpirrolidin-1-io-1-yl)but-2-inyl]fluoren-9-ol toblock the proliferation of the T cells that respond to the presence ofthe antigenic peptide 261-273 (huCollp261) only in the context ofHLA-DR4 or DR7 that broadly share the ability to bind peptides.

While limited to this experiment the two other molecules tested (1S and3S) show more modest inhibition capacity and specificity, in anexperiment performed on a larger group of patients, the results of whichare reported in Table I, 1S and 3S also show a significant ability toinhibit the proliferation of T cells specific to Type II collagen in theDR4+ context.

TABLE I PBMC from 4 patients with RA HLA-DR4+ are stimulated withhuCollp261 in the presence of each of the 3 compounds that are theobject of this patent. Number of specific Number of cells Compound Tcells inhibited % 1S 15 2 13 2S 15 9 60 3S 11* 5 45 *in one patient itis not possible to test this compound due to the insufficient number ofcells obtained from sampling.

Example 4 Test for Proliferation of T Cells in DR1+ Patients

Three types of T cells specific to huCollp262 (TRBV25 (133), TRBV19(101) and TRBV25 (146)), obtained from a patient Dr1+ DR4−, aresubjected to a proliferation test conducted as described in the previousexample 2. In particular, the test is performed in the absence ofantigen (ag−), in the presence of antigen (ag+) and in the presence ofag+ and of a candidate inhibitor compound (1S and 2S). The results areexpressed as a % of inhibition of T cell expansion (TRBV25 (133), TRBV19(101) and TRBV25 (146)) in the absence of inhibitor, at the indicatedconcentrations of the two inhibitors. As shown in FIG. 2, the compound1S, i.e.((E)-3-(3,6-dichloro-9H-carbazol-9-yl)-N-(4-hydroxy-benzylidene)propanidrazide) is able to strongly depress (about 100%) the Rate ofSpecific Stimulation (RSI) of T cells in the DR1+ patient, in two cellsout of three at a concentration 10 times lower than the compound 2S(9-[4-(3(R)-phenyl-1(S)-propilpirrolidin-1-io-1-yl)but-2-ynyl]fluoren-9-ol).These data demonstrate therefore that the compound 1S is a specificinhibitor of the recognition of collagen II, also in the context ofDR1+.

1-15. (canceled)
 16. An inhibitor of interaction between T cells and acomplex of MHC-II and human collagen type II or an antigenic fragmentthereof, for use in the treatment of rheumatoid arthritis, characterizedin that said inhibitor has the chemical formula of formula (II):

in which R and R¹ are independently selected from the group consistingof hydrogen, chlorine, bromine and iodine; R² is a substituent selectedfrom the group consisting of hydroxyl, methyl, ethyl, n-propyl, andisopropyl; R³ is N or CH; R⁴ is N or CH; and is in the form of a racemicmixture of isolated enantiomers or their salts; or formula (I):

in which R is selected from the group consisting of hydrogen, a methyl,ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, phenylethylgroup, and substituted o-chloro, o-bromo or o-iodo forms thereof; R1 isselected from the group consisting of: phenyl, 2′-pyridyl, 3′-pyridyl,4′-pyridyl, o-iodophenyl, m-iodophenyl, p-iodophenyl, o-chlorophenyl,m-chlorophenyl, and p-chlorophenyl; and is in the form of adiastereoisomeric mixture or racemic mixture or isolated enantiomer ortheir salts; or formula (III):

in which R and R¹ are independently selected from the group consistingof hydrogen, chlorine, bromine, and iodine; R² is selected from thegroup consisting of O, NH, and CH₂; and R³ is selected from the groupconsisting of O, S, and salified forms thereof.
 17. The inhibitoraccording to claim 16 for use in the treatment of rheumatoid arthritis,in which the antigenic fragment of human collagen type II is the peptide261-273.
 18. The inhibitor according to claim 16, for use in thetreatment of rheumatoid arthritis, in which the inhibitor is selectedfrom the group consisting of compounds 1 to 15 of formula (I), compounds1 to 15 of formula (II), and compounds 1 to 15 of formula (III).
 19. Theinhibitor according to claim 18, for use in the treatment of rheumatoidarthritis, selected from the group consisting of9-[4-(3(R)-phenyl-1(S)-propilpirrolidin-1-io-1-yl)but-2-ynyl]fluoren-9-ol;(E)-3-(3,6-dichloro-9H-carbazol-9-yl)-N′-(4-hydroxy-benzylidene)propanidrazide, N1,N5-bis(dibenzo[b,d]furan-3-yl) glutarammide, andtheir salts.
 20. A method for treating rheumatoid arthritis, comprisingadministering an inhibitor according to claim 16 to a patient who isHLA-DR4 and/or HLA-DR7 positive.
 21. A pharmaceutical compositionincluding, as active ingredient, a compound according to claim 16 and apharmaceutically acceptable excipient.
 22. A method for treatingrheumatoid arthritis, comprising administering a composition accordingto claim
 21. 23. A compound of general formula (II)

or salts thereof selected from the group consisting of:

or general formula (I)

in the form of a diastereoisomeric mixture (RR)/(SS) or (RS)/(SR) or ofan individual racemic mixture or an individual enantiomer (RS), (SR),(RR), (SS), or salts thereof selected from the group consisting of:

or general formula (III)

or salts thereof selected from the group consisting of:


24. A method for preparing a compound of formula (II), comprising atleast one of the steps listed in synthesis scheme 2:

or a compound of formula (I), comprising at least one of the stepslisted in synthesis scheme 1:


25. The method according to claim 24 further comprising resolvingdiastereoisomeric mixture (RR)/(SS) and (RS)/(SR) into individualracemic mixtures or resolving individual enantiomer (RR), (SS), (RS), or(SR).